Pharmaceutical, biotechnology, or genomics companies use DNA analysis systems for target identification and drug screening in pharmaceutical drug discovery. In many of these systems, biomolecules (e.g., DNA, RNA, cDNA, proteins) labeled with various dyes bind to chips that offer different molecular probe counterparts for binding in different locations of the chip. A scanner is then used to read the fluorescence of these resultant surface bound molecules under illumination with suitable (most often laser) light. The scanner acts like a large field fluorescence microscope in which the fluorescent pattern caused by binding of labeled molecules is scanned on the chip. In particular, a laser induced fluorescence scanner provides for analyzing large numbers of different target molecules of interest, e.g., genes/mutations/alleles, in a biological sample.
The scanning equipment typically used for the evaluation of arrays includes a scanning fluorimeter. A number of different types of such devices are commercially available from different sources, such as Axon Instruments in Union City, Calif. and Perkin Elmer of Wellesly, Mass. Analysis of the data, (i.e., collection, reconstruction of image, comparison and interpretation of data) is performed with associated computer systems and commercially available software, such as GenePix by Axon Instruments, QuantArray by Perkin Elmer or Feature Extraction by Agilent of Palo Alto, Calif.
In such scanning devices, a laser light source generates a—most often collimated—beam. The collimated beam sequentially illuminates small surface regions of known location on an array substrate. The resulting fluorescence signals from the surface regions are collected either confocally (employing the same lens used to focus the laser light onto the array) and/or off-axis (using a separate lens positioned to one side of the lens used to focus the laser onto the array). The collected signals are then transmitted through appropriate spectral filters to an optical detector. A recording device, such as a computer memory, records the detected signals and builds up a raster scan file of intensities as a function of position, or time as it relates to the position. Such intensities, as a function of position, are typically referred to in the art as “pixels” or “pixel values.”
If the expected or intended position of the feature is sufficiently close to its true position and laser intensity/detector sensitivity is set appropriately, the pixels within a region centered upon the expected or intended position of a feature can be averaged to yield the relative quantity of target bound to the probe in that feature. However, a user often has little idea of the brightness of the fluorescence that will be emitted by a particular sample. Accordingly, where applicable, the user does not know a priori how high or low to set an attenuator that controls the optical excitation signal power, that is, the signal power that reaches the sample. Likewise, the user will not know how high or low to set the gain of a detector that collects emitted fluorescence and produces a corresponding data signal.
The most apparent value of proper scanner scale factor or sensitivity choice/setup has to do with the amount of reliable information that can be obtained from a scan. Setting laser power and/or detector sensitivity too low may result in failure to collect weak signal information.
Early techniques for adjusting an optical scanner's scale factor involve manually setting the sensitivity of the system, where a user adjusts both the gain of the fluorescence detector and attenuation of the excitation light source. Typically, the user manually scans a sample in raster fashion to locate an element in the micro-array that is known to contain a concentration of a fluorophore that should produce a maximum fluorescence in response to the excitation signal. The user then re-scans the portion of the sample that contains this element and iteratively adjusts the sensitivity of the system until, in the judgment of the user, the corresponding data signal is sufficiently close to a maximum data signal value of the system. If the system has two channels, that is, produces excitation signals using two lasers of different wavelengths, the user re-scans the sample using the signal produced by the second laser/excitation light source and repeats the iterative, manual adjustment process the second channel. A user would further re-scan the sample for each additional channel.
The adjustment ranges for the attenuator or excitation source power and the detector are relatively large. Accordingly, manual adjustment of these components is time consuming. Thus, with manual scanning, the sample may be scanned many times to set the sensitivity of, or calibrate, the system. When multiple channels are used, more time is spent manually calibrating the system and the sample is scanned even more times, as discussed above.
U.S. Pat. No. 6,078,390 to Bengtsson describes a scanning system and method of operation for automatically setting detection sensitivity. It employs an optical scanning system using a low-resolution scanning operation to automatically adjust the sensitivity of the system. The system performs a low-resolution scanning operation by scanning a line, automatically and iteratively setting the levels of excitation signal power and detector gain, skipping a plurality of lines and scanning a next line, adjusting the levels as appropriate, skipping a plurality of lines and scanning a next line, and so forth. After the system sensitivities have been set, the calibrated system then scans all the lines of the sample to actually collect data. The calibrated system thus scans for the first time the lines that were skipped during the low resolution “calibration” scanning operation.
For these skipped lines, photo-bleaching (i.e., weakening of fluorescent signal caused by exposure to excitation light) is avoided. With the other lines, however, the same problems encountered with manual scanning and tuning optical system attenuation or excitation system gain from photo-bleaching as a result of rescanning are encountered. The risk of damage to the sample is further increased when multiple channels are used.
Another approach to setting scanner sensitivity is represented in U.S. patent application Ser. No. 10/910,552, entitled, “Maximum Sensitivity Optical Scanning System,” to B. Curry, et al. Here, scans are conducted in an iterative fashion from a maximum sensitivity scan, to scan(s) of lesser sensitivity when saturated results are present. If no signal saturation is detected after completing a scan, one or more increased sensitivity scans may be conducted, to edge system sensitivity upward.
The present invention teaches another approach to setting scanner sensitivity. The approach advantageously offers a less iterative approach in which a quick rescan of features can be made without saturation for improved data acquisition. Further, the invention may be used to safeguard detectors from exposure at too high a signal over too long a period of time. In addition, various data processing options or features may be offered by the present invention.